Optimizing the risk stratification of astrocytic tumors by applying the cIMPACT-NOW Update 3 signature: real-word single center experience

Our work reports implementation of a useful genetic diagnosis for the clinical managment of patients with astrocytic tumors. We investigated 313 prospectively recruited diffuse astrocytic tumours by applying the cIMPACT-NOW Update 3 signature. The cIMPACT-NOW Update 3 (cIMPACT-NOW 3) markers, i.e., alterations of TERT promoter, EGFR, and/or chromosome 7 and 10, characterized 96.4% of IDHwt cases. Interestingly, it was also found in 48,5% of IDHmut cases. According to the genomic profile, four genetic subgroups could be distinguished: (1) IDwt/cIMPACT-NOW 3 (n = 270); (2) IDHwt/cIMPACT-NOW 3 negative (= 10); (3) IDHmut/cIMPACT-NOW 3 (n = 16); and 4) IDHmut/cIMPACT-NOW 3 negative (n = 17). Multivariate analysis confirmed that IDH1/2 mutations confer a favorable prognosis (IDHwt, HR 2.91 95% CI 1.39–6.06), and validated the prognostic value of the cIMPACT-NOW 3 signature (cIMPACT-NOW 3, HR 2.15 95% CI 1.15–4.03). To accurately identify relevant prognostic categories, overcoming the limitations of histopathology and immunohistochemistry, molecular-cytogenetic analyses must be fully integrated into the diagnostic work-up of astrocytic tumors.


Patient cohort
All patients prospectively admitted to the Neurosurgery department of our Regional Hospital and referred to the Molecular Medicine Laboratory (Department of Medicine and Surgery, University of Perugia), for molecularcytogenetic diagnosis, were included in the study (timeframe: from August 2010 to July 2020).Overall, 313 patients were recruited.There were 184 males and 129 females.The median age, at diagnosis, was 64 years (range 23-83); the median follow-up was 10.9 with a range of 0.2-168 months.Survival was measured from the date of histopathological diagnosis until death, or was censored at the date of last follow-up.Median follow-up was 18.3 months: 24.3 for DA/AA and 9.6 for GBM.
In all cases the backbone of treatment was based on alkylating agents and/or radiotherapy except for 41 patients with multifocal diffuse disease and compromised clinical conditions, who died within 3 months from diagnosis.Timing, dosing, and scheduling were determined based on age, Karnofsky performance status, size of residual tumour, and presence of multifocal lesions.Radiotherapy started within 3-5 weeks after surgery, at a dosage of 50-60 Gy in 1.8-2 Gy, daily fractions.In fit patients, aged less than 70 years, temozolomide was concomitantly administered at 75 mg/m 2 daily dosage, plus at least six cycles of maintenance (150-200 mg/ m 2 , 5 out of 28 days) 10 .Patients with DA/AA were treated with radiotherapy alone or in combination with alkylating agents (temozolomide, lomustine, or carmustine) 11,12 .Molecular cytogenetic studies were done on paraffin-embedded biopsies taken at the time of diagnosis.Analyses were performed on representative areas as indicated by Pathologists.

Statistical analysis
Descriptive statistics were calculated including frequencies, percentages, frequency tables for categorical variables, median and means ± standard deviation (SD) for quantitative variables.Categorical variables were evaluated by Chi-square or Fisher's exact test when appropriate.The Kaplan-Meier method was used to analyze Overall survival (OS) and estimate medians with two-sided 95% confidence intervals (CI).Survival curves were compared using the log-rank test.Cox regression model was applied to estimate Hazard Ratio (HR) and 95% CI and to identify prognostic factors independently associated with survival times.To test proportional hazard (PH) assumption log-minus-log plots was used 14 .Stepwise backward-selection was used for eliminates variables from the regression model to find a reduced model that best explains the data 15 .A p-value of less than 0.05 was considered to be statistically significant.Statistical analyses were performed with STATA v. 16.1 (StataCorp LP, College Station, TX, USA).

Ethics aproval and consent to particpate
This study was performed in line with the principles of the Declaration of Helsinki and it was approved by the Umbria region ethic committee, code number 2843/16 on August 8th, 2016.Informed consent, regarding data was obtained from all individual participants included in the study.

The genomic profile of glioma tumours
Cases were first classified on the basis of histopathological and immunohistochemistry features as GBM (276), AA (17), and DA (20) (Table 1) 2 .According to the status of IDH1/2 genes, there were 280 IDH wildtype (IDH wt ) cases, including 267 GBM, 8 DA, and 5 AA, and 29 IDH-mutant (IDH mut ), which harbored IDH1R132, IDH2R140, or IDH2R172 1 .They were 5 GBM, 12 DA, and 12 AA (Table 1).In addition, 4 cases had non-canonical IDH1 or IDH2 mutations (cases nos.3, 4, 6, and 9, Table 2).These mutations were all located within the hot-spot region (exon 4), were not reported as polymorphisms, and were predicted to be pathogenic/likely pathogenic and/or described as somatic in other tumor types (Table 2) [16][17][18][19] .Therefore, since cases with non-canonical IDH1/IDH2 mutations could not be definitively considered IDH wt , we grouped them with IDH mut cases, while not strictly applying the WHO criteria 1 .Altogether, in our case series a total of 33 cases were considered to be IDH mut  By applying the cIMPACT-NOW Update 3 signature, 270 cases were reclassified as "Diffuse astrocytic glioma, IDH-wt, with molecular features of glioblastoma, WHO grade IV" due to the presence of at least one of biomolecular markers that define the cIMPACT-NOW 3 signature.All 13 IDHwt DA/AA cases, 11 of whom had at least 2 high-risk cIMPACT-NOW markers and none had an isolated TERTp mutation, belonged to this subgroup.
The IDH status was confirmed to be a robust prognostic marker (IDH wt , HR 2.91 95% CI 1.39-6.06)distinguishing two relevant risk subgroups.We also validated the cIMPACT-NOW 3 signature as an additional prognostic marker which improved the stratification of IDH wt tumours (cIMPACT-NOW 3, HR 2.15 95% CI www.nature.com/scientificreports/1.15-4.03).Indeed, the probability of overall survival was significantly lower in cIMPACT-NOW 3 positive (high risk subgroup) than in IDH wt cIMPACT-NOW 3 negative tumours (low risk subgroup) (Fig. 1B).The 13 IDH wt DA/AA cases that belonged to the cIMPACT-NOW 3 high risk subgroup showed a median overall survival (8.8 months; range 10-47) similar to GBM cases, although the two medians were statistically different probably due to the limited number of these rare cases.In addition, 12/13 patients died from disease progression.Instead, the cIMPACT-NOW 3 signature did not appear to be prognostically relevant in IDH mut cases (Table 2).On the other hand, according to the overall survival, the cIMPACT-NOW 3 negative GBM cases, either IDH wt (Table 3) or IDH mut , could be probably relocated into a more favorable risk subgroup (median overall survival: 55.8 months (29.5-not reached).

Discussion
Our study, conducted on a prospectively recruited case series, has the strength to broadly represent the incidence and distribution of recurrent astrocytic tumors in adult subjects in the real world.Indeed, as expected in adults, the number of patients with low grade IDH mut tumors was rather Thus, we are aware that this type of study might cause a bias in the analysis of patient survival related to differences in treatment and underrepresentation of rare subtypes.On the other hand, the study precisely reflects the epidemiological features of astrocytic tumors in adults.In addition, as a long-term follow-up was available, the outcome of long-term survivors could be precisely determined.Overall, from our study further support the rationale for incorporating the cIMPACT-NOW 3 signature as diagnostic criteria for glioblastoma 1 .
We confirmed that both IDH status and cIMPACT-NOW 3 signature are valid prognostic markers for risk stratification of patients and also provided new insights to consider for reclassification of low-risk cases.
As expected, the IDH status was found to be a robust prognostic marker distinguishing two relevant risk subgroups.Then to determine the impact of the genomic profile on overall survival, we assessed how the cIM-PACT-NOW 3 signature was distributed into IDH wt and IDH mut cases.As expected, the large majority of IDH wt cases were characterized by high risk signature.In particular, 92.6% cases were positive for at least two markers, and TERTp mutations were detected in 90% cases.Interestingly, 10 (~ 3.5%) IDH wt GBM did not harbor the cIMPACT-NOW 3 signature.
Survival analysis clearly indicated that the cIMPACT-NOW 3 signature is an additional prognostic marker suitable to improve the risk stratification of IDH wt cases.In fact, the probability of overall survival was significantly lower in cases with the cIMPACT-NOW 3 signature than in cases without this high-risk marker.. Notably, all the 13 IDH wt DA/AA belonged to the cIMPACT-NOW 3 high risk subgroup and showed a median overall survival similar to GBM cases.This is probably due to the genomic profile of our IDHwt DA/AA cases, which never had the isolated TERTp variants and showed 2-3 high-risk markers.Similarly, Berzero et al. 22 reported that astrocytomas with 2-3 molecular traits of the cIMPACT-NOW 3 signature had a more severe prognosis.However, they also showed that high risk markers are unequally distributed among grade II and grade III astrocytoma and that isolated TERTp mutations were not predictive of poor outcome 22 .
Other groups have shown that glioma cases with gain of 7p, loss of 10q, and mutation in the TERT promoter biologically represent a different subtype, which is prognostically relevant and has clinical impact for proper risk stratification of patients and choice of treatment [23][24][25] .On the other hand, we and others have observed that IDHwt GBM without the cIMPACT-NOW 3 signature behaved as low risk gliomas showing a long survival.
These findings, based on data from real-world diagnostic activity, further highlight the inherent limitations of histopathology and immunohistochemistry in the classification of glial tumors, and indicate the need for comprehensive characterization to properly stratify patients at the time of diagnosis.They are in line with other studies that have shown that IDH wt astrocytomas are more likely to behave like GBM, even in the absence of a high-risk molecular profile 24,26 .However, to confirm these data, large multicenter prospective studies need to be conducted.
Thus, while the cIMPACT-NOW 3 signature, the IDH status and age, were all strong predictors of outcome, histopathology and immunohistochemistry lost their prognostic significance.By introducing genetics among the dignostic criteria, the fifth edition of the WHO classification 1 established that regardless of histopathology, cases with high-risk molecular markers should be classified as GBM IDHwt.Instead, according to the overall survival, GBM cases, either IDH wt or IDH mut , not harboring this high risk signature, could be relocated into a more favorable risk subgroup.
Although the cIMPACT-NOW 3 signature was recommended to refine the classification of IDH wt gliomas, we sought to assess whether it also occurred in cases with IDH1/2 mutations and if it impacts upon prognosis in this subgroup of gliomas.Unexpectedly, roughly 48% of IDH mut cases harbored cIMPACT-NOW 3 markers which however did not appear to be prognostically relevant in this subgroup of tumors.
In conclusion, as recommended by the last updated WHO classification for CNS tumors, to accurately identify relevant prognostic categories, overcoming the limitations of histopathology and immunohistochemistry, a comprehensive molecular-cytogenetic approach must be considered in the diagnostic work-up of this subgroup of human cancers.Remarkably, more than 20 genes/pathways have been proposed to refine the classification of specific nosological entities in the context of gliomas and astrocytic gliomas in children and adults 1 .Defining the genomic profile of glioma tumours is not only required to predict response to chemo-/radio-therapy and life expectancy of patients, but also to provide the molecular basis for tailored treatments.